Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During tumor progression, micrometastases at their earliest stages have been difficult to analyze qualitatively or quantitatively because of a lack of suitably sensitive markers to discriminate small numbers of tumor cells from normal tissue cell populations. To overcome this problem, the Escherichia coli beta-galactosidase (lacZ) gene was introduced into human EJ Ha-ras oncogene-transfected BALB/c 3T3 cells with subsequent injection of transfected cells into athymic nude mice. Using a chromogenic substrate (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside), the lacZ-bearing tumor cells at primary tumor sites as well as at secondary organs stain intensely blue and can be easily distinguished from the host tissue cells hours, days, or weeks postinjection. Staining of lacZ-bearing tumor cells is specific and extremely sensitive in detecting micrometastatic foci in lungs and other organs, including brain and kidney for the first time. Stable integration of the lacZ and ras genes into cultured cells and subsequent tumor cells was verified by Southern blot analyses. The lacZ gene appears to be a stable marker during tumor progression in vivo based both on phenotypic (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside staining) and on genotypic (Southern blot analysis) evidence. Furthermore, 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside staining of tumor cells can also be used together with alkaline phosphatase staining relatively specific for endothelial cells to relate the topographies of metastatic cells and host blood vessels in embedded sections. By using the lacZ gene as a sensitive quantitative marker, analyses of micrometastasis development in the lung indicate that the ras oncogene contributes to the metastatic phenotype in this EJ Ha-ras model system, although further genetic and/or phenotypic alterations appear to be necessary for long-term growth and development into overt metastases. These findings demonstrate the effectiveness and sensitivity of the bacterial lacZ gene as a phenotypic marker in tumor progression studies, providing both a qualitative and a quantitative tool in virtually any tumor system for examining micrometastasis formation in target organs and the relationship of tumor cells to host organ microenvironments.
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PMID:Bacterial lacZ gene as a highly sensitive marker to detect micrometastasis formation during tumor progression. 218 31

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.
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PMID:Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A. 226 2

This study describes a simple and rapid method for the preparation of brush-border membrane vesicles from intestinal biopsies. The specific activities of sucrase, amino peptidase N, and alkaline phosphatase in these vesicles were the same as those in vesicles prepared from intestinal segments. The vesicles from all the regions of the small intestine can transport D-glucose in an Na+-dependent manner. The rates of transport of D-glucose presented here are far higher than previously reported. The method should have a wide applicability to studies of transport mechanisms and the distribution of transport processes within the intestine.
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PMID:Preparation and properties of brush-border membrane vesicles from human small intestine. 229 71

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.
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PMID:Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose. 232 32

We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic carcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subunit)-labelled ganglioside GM1 (diffusion coefficient, D [x 10(8)] = 0.8-0.9 cm2s-1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 10(9)] = 2 cm2s-1; R = 60-70%). However, antibody-labelled beta 2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was x 1.4 and R x 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were x0.60 and x0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobility of different membrane components.
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PMID:Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA class 1, HLA class 2, and neoplastic epithelial antigens) in the apical cell membrane. 233 58

In an open, exploratory study, the safety of ursodeoxycholic acid (UDCA) in the treatment of primary biliary cirrhosis (PBC) was investigated. Seven patients in stages I to III and two patients in stage IV were treated for 1 year with 1 g/day of UDCA. Clinical symptoms, and alkaline phosphatase, gamma-glutamyltransferase, alanine aminotransferase (GOT) and aspartate aminotransferase (GTP) levels improved significantly within three months and remained at the lower levels for the period of observation. Results of the galactose elimination capacity (4.7 +/- S.D. 1.4 mg/min per kg) and the aminopyrine breath test (0.60 +/- 0.33% dose/kg per mmol CO2) remained unchanged for 1 year. In all patients total serum bile acids increased and quantitatively UDCA became the most important bile acid. In patients in stages I to III this increase, however, was modest, whereas in patients in stage IV, total serum bile acids reached levels of 140 and 157 mumol/l and UDCA, levels of 90 and 103 mumol/l, respectively. It is concluded that UDCA appears to be safe only in stages I to III and that prognostic stratification based on bile acid levels or on the histological stage of the disease should be an important aspect of controlled clinical trials.
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PMID:Ursodeoxycholic acid in primary biliary cirrhosis: no evidence for toxicity in the stages I to III. 236 81

The prevalence of biliary and hepatic diseases is increasing in patients with cystic fibrosis as more of them reach adult life. There is no effective treatment or method of preventing cholestasis in cystic fibrosis, although beneficial effects have been ascribed to the tertiary bile acid, ursodeoxycholate, in other forms of chronic cholestasis. We evaluated prospectively the effects of a six month course of ursodeoxycholate (15-20 mg/kg per day) in eight, mostly adult, patients with cystic fibrosis and chronic cholestasis. Bile acid treatment improved inflammatory activity (average decrease in alanine aminotransferase, 60%, p less than 0.005) and cholestasis (alkaline phosphatase, 47%; p less than 0.01) in all patients. Quantitative liver function, measured by 45 minute sulphobromophthalein retention and by the 14C-aminopyrine breath test, improved in all patients while galactose elimination capacity showed a slight decrease. Patients' nutritional state improved as evidenced by a 1.8 kg weight gain and an increase in muscle mass suggested by a 26% increase in 24 hour urinary creatinine excretion. Steatorrhea was not affected by bile acid treatment. Ursodeoxycholic acid may be beneficial in the treatment of chronic cholestasis in cystic fibrosis by improving liver function and also the patient's nutritional state.
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PMID:Effects of ursodeoxycholic acid treatment on nutrition and liver function in patients with cystic fibrosis and longstanding cholestasis. 238 18

A simple and sensitive chromogenic microtitre plate assay for glycoprotein enzymes is described, using melanoma tissue plasminogen activator (t-PA) as a model enzyme. The assay is based on the binding of t-PA to immobilised lectin and quantitating the bound enzyme with plasminogen, fibrinogen fragments and chromogenic substrate S-2251 on an ELISA plate reader. Seven different lectins were examined for the binding of t-PA, and of these, concanavalin A was chosen for subsequent studies. The specificity of this binding can be inhibited dose-dependently in the presence of D-mannose and methyl alpha-D-mannoside, but not by D-glucose and D-lactose. The lower limit of the sensitivity of this assay is about 0.5 IU/ml. Comparison of the dose-response curves indicates that the sensitivity of this assay method is very similar to that of bioimmunoassay using anti-t-PA IgG to capture the antigen. The applicability of this method to other glycoprotein enzymes was also evaluated using alkaline phosphatase from bovine mucosa. The specificity of this method was related to the choice of substrate and this was shown by analysis of a mixture of t-PA and alkaline phosphatase. It is suggested that this assay can be adapted for the analysis of complex glycoprotein mixtures with the appropriate choice of lectin and substrate.
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PMID:Lectin affinity bioassay: an assay method for glycoprotein enzyme. 249 37

In the presence of inhibitors for mitochondrial H+-ATPase, (Na+ + K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N.N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brush-border and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator. ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl-) or entry of a cation, e.g., Na+ via electrogenic Na+/D-glucose and Na+/L-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM- sensitive ATPases at the cytosolic side of the brush-border membrane.
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PMID:Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion. 253

Three clones of the pig kidney cell line LLC-PK1 were isolated and characterized with regard to morphology, growth, proximal tubule enzyme activity, sugar uptake capacity, and hormone and drug responsiveness in a defined medium. Clone N4 was similar in morphology to the wild type (WT), whereas clone F8 showed loose attachment to the substrate, formed large, sweeping domes, and had an elongated desmosome junction between cells. The third clone, F2, did not form domes and showed a marked reduction in growth rate. Cultures of WT, N4, and F8 had higher specific activities of the enzyme alkaline phosphatase and gamma-glutamyl transpeptidase at confluence relative to growing cells; however, there was no evidence of an increase in activity of either enzyme at confluence in F2. Phlorizin-sensitive alpha-methyl-D-glucoside uptake and cytochalasin B-sensitive 2-deoxy-D-glucose uptake were measured in confluent cultures grown on porous filter supports. None of the clones lacked either of the hexose transport systems, although quantitative differences were evident. N4 cells grown in a defined medium in 96-well culture plates were tested in situ for their enzyme responses to differentiation inducers, tumor promoters, and hormones. Alkaline phosphatase activity was significantly increased at confluence by serum, parathyroid hormone (PTH), and vasopressin (AVP), and was decreased by tetradecanoylphorbol acetate (TPA) and epinephrine (EPI). Glutamyl transpeptidase activity was decreased at confluence by serum, TPA, and EPI. Similar tests on alpha-methyl-D-glucoside uptake showed that serum, TPA, PTH, and AVP had no significant effect on phlorizin-sensitive uptake; however, calcitonin increased uptake by 84% (n = 18). It was concluded that LLC-PK1 clones maintained in a defined medium are useful models for studying renal cell function.
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PMID:Growth, enzyme activity, sugar transport, and hormone supplement responses in cells cloned from a pig kidney cell line LLC-PK1. 256 38


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